Abstract
Mammalian voltage-gated K+ channels are assemblies of pore-forming alpha-subunits and modulating beta-subunits. To operate correctly, Kv4 alpha-subunits in the heart and central nervous system require recently identified beta-subunits of the neuronal calcium sensing protein family called K+ channel-interacting proteins (KChIPs). Here, Kv4.2.KChIP2 channels are purified, integrity of isolated complexes confirmed, molar ratio of the subunits determined, and subunit valence established. A complex has 4 subunits of each type, a stoichiometry expected for other channels employing neuronal calcium sensing beta-subunits.
Highlights
In muscles and nerves, Kv4 (Shal family) ␣-subunits assemble with Kϩ channel-interacting proteins (KChIPs)1 -subunits to create mixed complexes with unique attributes and functions [1,2,3,4,5,6,7]
To explore the structural basis for KChIP function, the stoichiometry of Kv4.21⁄7KChIP2 channels was determined using a stepwise strategy: significant amounts of intact, functional Kv4.21⁄7 KChIP2 complexes were produced and isolated; functional and structural integrity of the purified material was confirmed by a toxin binding assay; and, thereafter, subunit valence was established by gradient centrifugation and direct amino acid analysis
We demonstrate that regulatory KChIP2 subunits assemble with pore-forming Kv4.2 subunits in 4:4 complexes when over-expressed in tissue culture cells to produce voltage-gated Kϩ channels like those in native cells (such as cardiac Ito [3, 4])
Summary
Kv4 (Shal family) ␣-subunits assemble with Kϩ channel-interacting proteins (KChIPs)1 -subunits to create mixed complexes with unique attributes and functions [1,2,3,4,5,6,7]. KChIPs (and frequenin) [16, 17] have been shown to assemble with Kv4 subunits in stable fashion leading to increased current density due to enhanced surface expression, activation at more hyperpolarized potentials, slowed inactivation, and speeded recovery from inactivation [1, 2, 16]. These -subunits permit kinase regulation [18], control trafficking, and alter surface half-life [6]. To explore the structural basis for KChIP function, the stoichiometry of Kv4.21⁄7KChIP2 channels was determined using a stepwise strategy: significant amounts of intact, functional Kv4.21⁄7 KChIP2 complexes were produced and isolated; functional and structural integrity of the purified material was confirmed by a toxin binding assay; and, thereafter, subunit valence was established by gradient centrifugation and direct amino acid analysis
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
